Butirosin A 3",5"-O-isopropylidene derivatives

Tetra-N-benzyloxycarbonylbutirosin A (2)*

To a stirred mixture of 19.5 g. (35 mmoles) of butirosin A (1) and 8.20 g. (77.3 mmoles) of Na 2 CO 3 in 440 ml. of 20% aqueous acetone was added dropwise 26.2 g. (153 mmoles) of benzyl chloroformate under cooling. The reaction mixture was stirred overnight at room temperature to give an oily precipitate, which was separated from the supernatant by decantation, washed thoroughly with water and triturated with ether to give 34.23 g. (89%) of 2.

Tetra-N-benzyloxycarbonyl-3',4';3",5"-di-O-isopropylidenebutirosin A (4)

A solution of 14.3 g. (13.1 mmoles) of 2, 30 ml. of 2,2-dimethoxypropane and 60 mg. of p-toluenesulfonic acid in 250 ml. of DMF (dimethylformamide) was allowed to stand at room temperature for 2 days, then evaporated in vacuo to remove the MeOH formed. The concentrate was treated with another 10 ml. of 2,2-dimethoxypropane, kept at 60°C. for 2 hours, treated with about 1 ml. of Et 3 N and evaporated to dryness in vacuo. The oily residue was chromatographed on a silica gel column (180 g.) and eluted with CHCl 3 -MeOH to give 5.5 g. (36%) of the diacetonide 4 and 8.45 g. (57%) of the monoacetonide 3*. Repeated isopropylidenation of 3 gave additional amount of 4. Total yield of 4 was 11.0 g. (72%). Monoacetonide (3), m.p. 123°-125°C.:

Anal. Calcd. for C 56 H 69 N 5 O 20 .sup.. 1/2 H 2 O: C, 58.93; H, 6.18; N, 6.14. Found: C, 58.98; H, 5.99; N, 6.09. Diacetonide (4), m.p. 121°-123°C.:

Anal. Calcd. for C 59 H 73 N 5 O 20 : C, 60.54; H, 6.28; N, 5.97. Found: C, 60.44; H, 6.26; N, 5.79. ##SPC24##

2",6,α-Tri-O-acetyl-tetra-N-benzyloxycarbonyl-3',4'; 3",5"-di-O-isopropylidenebutirosin A (5)

To a solution of 226 mg. (0.193 mmoles) of 4 in 10 ml. of dry pyridine was added 1.5 ml. of Ac 2 O. The reaction mixture was allowed to stand overnight and poured onto ice-water. The mixture was extracted with CHCl 3 . The CHCl 3 extracts were washed with water, dried with Na 2 SO 4 and evaporated in vacuo to give 248 mg. (99%) of 5. An analytical sample was obtained by chromatography on silica gel; m.p. 108°-110°C.

Anal. Calcd. for C 65 H 79 N 5 O 23 : C, 60.13; H, 6.13 N, 5.39. Found: C, 60.09; H, 6.09; N, 5.44.

2",6,α-Tri-O-acetyl-tetra-N-benzyloxycarbonylbutirosin A (6)

A solution of 2.0 g. (1.54 mmoles) of 5 in 15 ml. of 80% aqueous acetic acid and 15 ml. of acetone was heated at 80°C. for 3 hours and then evaporated to dryness in vacuo. The residue was chromatographed on a silica gel column (62 g.) with CHCl 3 -MeOH as eluent to give 1.55 g. (81%) of 6; m.p. 105°-106°C.

Anal. Calcd. for C 59 H 71 N 5 O 23 : C, 58.17; H, 5.87; N, 5.73. Found: C, 58.49; H, 6.18; N, 5.80.

2",6,α-Tri-O-acetyl-tetra-N-benzyloxycarbonyl-3",5"-O-isopropylidenebutirosin A (7)

To a solution of 1.82 g. (1.49 mmoles) of 6 in 24 ml. of dry DMF were added 24 ml. of 2,2-dimethoxypropane and 8 mg. of p-toluenesulfonic acid. The reaction mixture was allowed to stand overnight at room temperature, then treated with 0.1 ml. of Et 3 N, and evaporated to dryness in vacuo. The residue was purified on a silica gel column (30 g.) with CHCl 3 -MeOH to give 1.79 g. (96%) of 7; m.p. 105°-107°C.

Anal. Calcd. for C 62 H 75 N 5 O 23 : C, 59.18; H, 6.01; N, 5.57. Found: C, 59.37; H, 5.98; N, 5.41.

2",6,α-Tri-O-acetyl-tetra-N-benzyloxycarbonyl-3",5"-O-isopropylidene-3'-O-tosylbutirosin A (8)

To a stirred solution of 1.2 g. (0.955 mmoles) of 2",6,α-tri-O-acetyl-tetra-N-benzyloxycarbonyl-3",5"-O-isopropylidenebutirosin A (7) in 15 ml. of dry pyridine was added 900 mg. (4.72 mmoles) of tosyl chloride below -10°C. The reaction mixture was stirred for 2 days at room temperature and, after adding 10 ml. of water, evaporated to dryness in vacuo. The residue was triturated with water and dried in vacuo to yield 1.297 g. of crude 8, which was purified on a silica gel column (210 g.) with CHCl 3 --MeOH system to give 641 mg. (48%) of 8; m.p. 105°-106°C. IR (KBr): ν SO .sbsb.2 1175 cm - 1 .

Anal. Calcd. for C 69 H 81 N 5 O 25 S: C, 58.67; H, 5.78; N, 4.96; S, 2.27. Found: C, 58.83; H, 5.68; N, 4.77; S, 2.25.

3'-Deoxybutirosin A. BB-K186 (11)

7A.: A mixture of 282 mg. (0.2 mmoles) of 8, 520 mg. of NaI and 10 ml. of dry acetone in a sealed glass tube was heated at 120°-130°C. for 2 days, during which time crystals of sodium tosylate precipitated. The reaction mixture being filtered, the filtrate was evaporated to dryness in vacuo to leave an oily residue, which was washed with water and dried to give the 4'-iodo derivative (9) showing no ν SO .sbsb.2 band in its ir spectrum. 7B.: A solution of the iodo derivative in 10 ml. dioxane was hydrogenated with 5 ml. of Raney nickel (a thick suspension in dioxane) and hydrogen at 42 psi for 2 days in a Paar hydrogenator. The catalyst was removed by filtration and the filtrate was repeatedly hydrogenated overnight with 5 ml. of Raney nickel. The reaction mixture was filtered and the filtrate was dried up in vacuo. The yellowish residue (152 mg.) was dissolved in 10 ml. of 10 % NH 3 in MeOH, allowed to stand for 1.5 hours at room temperature and then dried up in vacuo. The deacetylated product was dissolved in 10 ml. of 50% aqueous AcOH, heated at 80°C. for 2 hours and evaporated to dryness in vacuo to give 103 mg. of tetra-N-benzyloxycarbonyl-3'-deoxybutirosin (10). 7C.: A solution of 10 in 15 ml. of 50% EtOH was hydrogenated overnight with 100 mg. of 20% Pd-C and 0.1 ml. of Et 3 N in a Paar apparatus at 42 psi. The catalyst was removed by filtration. The filtrate was evaporated to dryness in vacuo. The residue was chromatographed on a CG-50 column (NH 4 + form, 10 ml.) with 200 ml. of 0.1 N NH 4 OH, 200 ml. of 0.2 N NH 4 OH and 200 ml. of 0.5 N NH 4 OH as eluent. Each 10 ml. fraction was collected. Fractions showing Rf 0.35 on TLC (developed twice with S-110 system* 1 ) were pooled, evaporated to dryness in vacuo and lyophilized to give 12 mg. (11% from 8) of 11.

Amberlite CG-50 is the tradename for the chromatographic grade of weakly acidic cationic exchange resin of a carboxylic-polymethacrylic type.

Preparation of the monosulfate salt of 3'-deoxybutirosin A (11)

One mole of 3'-deoxybutirosin A is dissolved in 1 to 3 liters of water. The solution is filtered to remove any undissolved solids. To the chilled and stirred solution is added one mole of sulfuric acid dissolved in 500 ml. of water. The mixture is allowed to stir for 30 minutes, following which cold ethanol is added to the mixture till precipitation occurs. The solids are collected by filtration and are determined to be the desired monosulfate salt.

Preparation of Disulfate Salt of 3'-deoxybutirosin A (11)

Thirty-five grams of 3'-deoxybutirosin A is dissolved in 125 ml. of deionized water. The pH is adjusted to 7-7.5 with 50% V/V sulfuric acid.

Eight and one half grams of Darco G-60 (activated charcoal) is added and the mixture is slurried at ambient room temperature for 0.5 hour. The carbon is removed by suitable filtration and washed with 40 ml. of water. The water wash is added to the filtrate.

The combined filtrate-wash above is adjusted to pH 2-2.6 with 50% V/V sulfuric acid.

Eight and one half grams of Darco G-60 is added to the solution. The mixture is slurried for 0.5 hour at ambient room temperature. The carbon is removed by suitable filtration and washed with 35 ml. of deionized water. The water is added to the filtrate.

The combined filtrate-wash is adjusted to pH 1-1.3 with 50% V/V sulfuric acid. This solution is added with rapid stirring over a 10 minute period to 600-800 ml. of methanol (3-4 volumes of methanol). The mixture is stirred for 5 minutes at pH 1-1.3, passed through a 100 mesh screen, stirred for 2 minutes and allowed to settle for 5 minutes. Most of the supernatant is decanted. The remaining slurry is suitably filtered, washed with 200 ml. of methanol and vacuum dried at 50°C. for 24 hours to yield the desired disulfate salt of 11.

3'-Deoxybutirosin A

Substitution in the procedure of Example 7, in step 7A, for the sodium iodide used therein of an equimolar quantity of lithium bromide produces the compound having the formula ##SPC25##

Alternative preparation of 3'-deoxybutirosin A, BB-K186 (11)

Step A -- To a solution of 144 mg. of 2",6,α-tri-O-acetyl-tetra-N-benzyloxycarbonyl-3",5"-O-isopropylidene-3'-O-tosylbutirosin A (8) in 10 ml. of dry acetone was added 230 mg. of potassium thiolacetate and the mixture was heated in a sealed tube at 120°C. overnight. The sealed tube was cooled to room temperature and opened. The reaction mixture was filtered and the filtrate was evaporated in vacuo to dryness. The oily residue was triturated with water to give 121 mg. of the 3'-acetylthio derivative (12) which was purified by a column of silica gel (10 g.).

Step B -- A solution of the 3'-acetylthio derivative in 5 ml. of dioxane was treated with 3 ml. of Raney nickel (a thick suspension in dioxane) and the mixture was refluxed for 3 hours. The reaction mixture was filtered and the filtrate was evaporated in vacuo to dryness. The residue was dissolved in 30 ml. of 10% ammonia in methanol and kept to stand at room temperature overnight. The reaction mixture was evaporated in vacuo to dryness and the residue was heated at 80°C. for 1 hour with 25 ml. of a mixture of 20% aqueous acetic acid and acetone (1 : 1) to afford tetra-N-benzyloxycarbonyl-3'-deoxybutirosin (10), which was identical with the authentic sample prepared by iodination of 8 followed by hydrogenation with Raney nickel.

Step C -- A solution of 10 in 10 ml. of 60% aqueous ethanol was hydrogenated in the presence of 100 mg. of 20% Pd-C to give 5 mg. of BB-K186 (11), which was identical with the authentic sample from Example 7.